Medicinal plant extracts are of interest as sources of novel antiviral agents. Melissa officinalis, commonly known as Lemon Balm, is an abundant botanical historically claimed to have strong antiviral properties. Melissa has a long medicinal history being used first up to two thousand years ago by the Greeks. Melissa is purported to help significantly in the treatment of herpes simplex viruses, HSV1 and HSV2.
The antiviral activity associated with Melissa has utilized glycerine-based extracts prepared from dried Melissa officinalis. The botanical was ground to a fine powder followed by resuspension in 75% glycerin (pharmaceutical grade, organic vegetable glycerin) at a ratio of 1:8 (dried plant material to extraction solution). The solution was stored at room temperature for seven days followed by removal of the botanical debris by centrifugation and sterilization through a 0.2 um filter.
Previous studies have demonstrated that extracts from Melissa officinalis can inhibit the replication of herpes viruses. As shown in FIG. 1 (lower left corner), cells infected with HSV lead to dramatic cytopathic effects and cell death. As cells were treated with increasing concentrations of Melissa extract, a dose dependent reduction in cell death was observed. Uninfected cells (FIG. 1, upper row) remained morphologically normal and healthy.
Many different cell surface molecules can serve as receptors for the attachment and entry of viruses. The particular receptor or receptors a virus can use will determine the cell types it can infect. For HSV1, cell surface heparin sulfate proteoglycans serve as the cell surface receptor. Previous data demonstrates that cells treated with heparinases or altered by mutations that prevent heparin sulfate biosynthesis have reduced capabilities to bind HSV1 and are partially resistant to virus infection. Soluble heparin has been shown to competitively inhibit HSV1 infection. Through temporal studies, it has been demonstrated that extracts of Melissa can inhibit HSV1 binding to cells. In support of this, since herpes viruses are known to bind to cells utilizing herparin sulfate proteoglycans, the ability of the Melissa extract to competitively inhibit herpes virus binding to a heparin-agarose resin was tested. As shown in FIG. 2, the Melissa extract blocked herpes virus binding to the resin as detected by a decrease in the viral glycoprotein B (gB). These and previous results suggest that Melissa acts by inhibiting herpes virus cellular attachment.
Blocking HSV1 attachment to cells could occur by components in Melissa either binding directly to the virus or by binding to the cell. As shown in FIG. 3, when cells were preincubated with the extract, washed and then infected with HSV1, virus replication was not inhibited. However, if the extract was preincubated with purified HSV1 virions, which were then washed and tested for infectivity, virus replication was inhibited (FIG. 3). These results support that the active constituent(s) in the Melissa extract bind to the herpes virion and inhibit viral attachment to the cell. The HSV1 glycoprotein B (gB) is one of the major viral surface proteins involved in binding the virus to heparin sulfate proteoglycans on the cell surface. Since Melissa components bind to the virus and inhibit binding to the cellular heparin sulfate proteoglycan, the HSV1 gB is a likely target for Melissa interaction. In support of this, the Melissa extract was depleted of gB-binding components by affinity chromatography. After removal of gB binding components present in the Melissa extract, the HSV1 inhibitory activity was greatly reduced (FIG. 4). This result suggests that the active constituent of Melissa binds to gB of the virion particle. In summary, these results support that Melissa extracts inhibit herpes virus binding to heparin sulfate on the cell surface by binding to the HSV1 gB and blocking viral attachment.
Cell cytotoxicity is an important concern when considering any potential therapeutic. In assays, inhibition of HSV1 virus replication occurred at a low dose (EC50=0.58 ul/ml) (FIG. 5). Upon testing for cell cytotoxicity, the extract did not exhibit detectable cell cytotoxicity with the CC50=190 ul/ml being comparable to glycerin (vehicle) alone. (FIG. 5).